Use of oxalate deficient aspergillus niger strains for producing a polypeptide

ABSTRACT

The invention relates to oxalate deficient  A. niger  strains for the production of a given enzyme, wherein the oxalate deficient strain produces at least the same amount of enzyme as the wild type strain it originates from under the same culture conditions. Preferably, the oxalate deficient  A. niger  strain produces more enzyme than the wild type strain it originates from under the same culture conditions. More preferably, the oxalate deficient  A. niger  strain is such that when the strain has been transformed with an expression construct comprising a gene coding for an enzyme, said strain produces at least the amount of the enzyme the wild type strain it originates from would produce under the same culture conditions, when the wild type strain has also been transformed with the same expression construct as the oxalate deficient strain. The invention also relates to method for obtaining such oxalate deficient  A. niger  strain. The present invention further relates to method for producing an enzyme, wherein an oxalate deficient  A. niger  strain that produces at least the same amount of enzyme as the wild type strain it originates from under the same culture conditions is used.

FIELD OF THE INVENTION

The invention relates to oxalate deficient Aspergillus niger strains forproducing a polypeptide, to their use and to a method for obtaining suchstrains.

BACKGROUND OF THE INVENTION

Oxalic acid is an undesirable by-product that accumulates in the culturesupernatant of cells during fermentation and causes difficulties in thedownstream processing of the desirable compound.

Four Russian prior art documents, named Ru1-Ru4 (defined hereafter),describe how to obtain oxalate deficient Aspergillus niger (A. niger)strains using classical mutagenesis methods. Oxalate deficient A. nigerstrains are defined as strains that produce less oxalic acid than theparental strain they originate from. They demonstrate that the choice ofthe mutagen agent is not critical: UV, or chemicals, or a combination ofboth as mutagen agents would lead to the obtention of oxalate deficientA. niger strains.

They use a chromatography assay to select strains that produce lessoxalic acid or more citric acid than the parental strain they originatefrom. They do not envisage to use these strains for producingpolypeptides.

Ru1: On methods of selecting A. niger mutants with altered capacity tosynthesize organic acids, ID Kasatkina and E. G. Zheltova,Mikrobiologiya, vol 34, no 3, p 511-518, May-June 1965.

Ru2: RU2089615, New strains of A. niger has properties of producer ofcitric acid and can be used in microbiological industry (DW1998-249164).

Ru3: The variability of A. niger, a producer of citric acid, under theinfluence of the separate and combined action of nitrosomethylurea andultraviolet rays, E. Y. Shcherbakova, Z. S. Karadzhova and V. P.Ermakova, Mikrobiologiya, vol 43, no 3, p 508-513, May-June 1974.

Ru4: Change in the ratio of citric acid and oxalic acids in A. nigerunder the influence of mutagenic factors, V. M. Golubtsova, E. Y.Shcherbakova, L. Y. Runkovskaya and V. P. Eramkova, Mikrobiologiya, vol48, no 6, p 1060-1065, November-December 1979.

Another publication, WO 00/50576 describes that oxaloacetate hydrolasedeficient host cells can be used for producing desirable compounds, suchas polypeptides, primary and secondary metabolites. These host cellshave less oxaloacetate hydrolase activity than the parental cells theyoriginate from. As a result, these oxaloacetate hydrolase (OAH)deficient cells produce less oxalic acid than the parental cells theyoriginate from. This patent application does not show experimental datademonstrating that an oxaloacetate hydrolase deficient cell is asuitable polypeptide producer. Furthermore, Pedersen et al, (Pedersen,H., et al, Metabolic Eng., (2000) 2, 34-41) later described thatoxaloacetate hydrolase deficient Aspergillus niger strains transformedwith a DNA construct comprising the DNA sequence encoding theglucoamylase enzyme are not able to produce the glucoamylase enzyme atthe level the wild type strain they originate from does under the sameculture conditions: the mutants produce 50% less glucoamylase than thewild type. Such a mutant is not suited as a polypeptide producer in anindustrial setting.

There is still a need for oxalate deficient A. niger strains that areable to produce at least the amount of a polypeptide a wild type strainwould produce and that can be used as polypeptide producer in anindustrial setting.

DETAILED DESCRIPTION OF THE INVENTION

Oxalate deficient A. niger strains suitable for the production of agiven polypeptide or enzyme in an industrial setting have been isolated,wherein surprisingly the oxalate deficient strain produce at least thesame amount of polypeptide or enzyme as the wild type strain theyoriginate from under the same culture conditions. Preferably, themutants produce at least the amount of polypeptide or enzyme the A.niger strain CBS 513.88 produces under the same culture condition.

In this application, A. niger strain CBS 513.88 is taken as a referenceof wild type oxalate levels obtainable in an A. niger culture, as areference of wild type polypeptide level obtainable in an A. nigerculture and as a reference of intracellular OAH activity obtainable inan A. niger culture. Oxalate deficient A. niger strains are defined asstrains that produce less oxalate than the A. niger strain CBS 513.88under the same culture conditions. Preferably, the oxalate deficient A.niger strains used produce no more than half the amount of oxalate thatthe wild type strain they originate from produces under the same cultureconditions. More preferably, the oxalate deficient A. niger strains usedproduce no more than one third of the amount of oxalate that the wildtype strain they originate from produces under the same cultureconditions. Most preferably, the oxalate deficient A. niger strains usedproduce no more than one fifth of the amount of oxalate that the wildtype strain they originate from produces under the same cultureconditions. More preferably, the oxalate deficient A. niger strains usedproduce no more than half the amount of oxalate that the A. niger strainCBS 513.88 produces under the same culture conditions. More preferably,the oxalate deficient A. niger strains used produce no more than onethird of the amount of oxalate that the A. niger strain CBS 513.88produces under the same culture conditions. Most preferably, the oxalatedeficient A. niger strains used produce no more than one fifth of theamount of oxalate that the A. niger strain CBS 513.88 produces under thesame culture conditions. According to a preferred embodiment of theinvention, the oxalate deficient A. niger strain used has been obtainedby applying the method defined later in this application.

Preferably, the oxalate deficient A. niger strains of the invention arestrains that produce more of a given polypeptide than the wild typestrain they originate from under the same culture conditions. Morepreferably, the oxalate deficient A. niger strain produces more of agiven polypeptide than the A. niger CBS 513.88 under the same cultureconditions.

A large variety of systems for detection of polypeptide are known to theskilled person. Detection systems include any possible assay fordetection of polypeptide or enzymatic activity. By way of example theseassay systems include but are not limited to assays based oncolorimetric, photometric, turbidimetric, viscosimetric, immunological,biological, chromatographic, and other available assays.

Preferably, if the polypeptide produced is an enzyme, the amount ofactive enzyme produced is determined by measurement of its activity in amodel reaction (see examples).

Preferably, the oxalate deficient A. niger strains of the invention arestrains having a detectable intracellular OAH activity as detected in amodel reaction (see experimental information in the Examples) Morepreferably, the oxalate deficient A. niger strains of the invention arestrains having an intracellular OAH activity, which is ranged between0.1 and 100% of the intracellular OAH activity of the wild type strainthey originate from as detected in a model reaction, preferably between0.5 and 90, more preferably between 0.5 and 80, even more preferablybetween 1 and 50, most preferably between 1 and 25 and even mostpreferably between 1 and 10. According to another preferred embodiment,the oxalate deficient A. niger strains have an intracellular OAHactivity, which is ranged between 0.1 and 100% of the intracellular OAHactivity of the CBS 513.88 deposited strain as detected in a modelreaction. More preferably, the oxalate deficient A. niger strains of theinvention are strains having an intracellular OAH activity, which isranged between 1 and 90% of the intracellular OAH activity of the CBS513.88 deposited strain as detected in a model reaction.

The existence of such oxalate deficient strains still having adetectable OAH activity, is surprising, since it was thought that OAHwas the only molecule responsible for the formation of oxalate. Mutantsstill having detectable level of OAH activity have several advantagescompared to oxalate deficient strains with no detectable OAH activity(Pedersen H et al, Metabolic Eng. (2000) 2, 34-41): they are able toproduce at least the amount of a given polypeptide the wild type strainwould produce under the same culture conditions. Furthermore, theendogenous metabolic pathway of organic acids is most likely notpertubated.

According to a further preferred embodiment, the oxalate deficient A.niger strain of the invention is characterized by the fact that whenthis strain has been transformed with an expression construct comprisinga gene coding for a polypeptide, said strain produces at least theamount of the polypeptide the wild type strain it originates from wouldproduce under the same culture conditions, when the wild type strain hasalso been transformed with the same expression construct as the oxalatedeficient strain.

The gene coding for the polypeptide to be produced may be homologous orheterologous to the oxalate deficient A. niger strain used. The term“heterologous” means that the polypeptide is not native to the A. nigercell. Preferably, the gene comprised in the expression construct is aheterologous gene for A. niger.

Preferred heterologous polypeptide is human serum albumine, lactoferrin,chymosin or Phospholipase A2. According to a preferred embodiment of theinvention, the oxalate deficient strain has been transformed with a DNAconstruct comprising a DNA sequence encoding said polypeptide.Preferably, the polypeptide is an enzyme. Enzymes that can be producedare carbohydrases, e.g. cellulases such as endoglucanases, β-glucanases,cellobiohydrolases or β-glucosideases, hemicellulases or pectinolyticenzymes such as xylanases, xylosidases, mannanases, galactanases,galactosidase, rhamnogalacturonases, arabanases, galacturonases, lyases,or amylolytic enzymes; phosphatases such as phytases, esterases such aslipases, proteolytic enzymes, oxidoreductases such as oxidases,transferases, or isomerases. Preferably, the amylolytic enzyme to beproduced is an alpha amylase (EC 3.2.1.1., alpha-1,4-glucan-4-glucanohydrolase or EC 3.2.1.2). More, preferably, the DNA sequence encodes afungal alpha amylase. Most preferably, the DNA sequence encoding thefungal alpha amylase is derived from A. niger or Aspergillus oryzae.According to another embodiment, the enzyme to be produced is a prolinespecific endoprotease (EC 3.4.16.2). According to another embodiment,the enzyme to be produced is a phospholipase A1 (PLA1) (EC 3.1.1.32).More, preferably, the DNA sequence encodes a fungal PLA1. Mostpreferably, the DNA sequence encoding the fungal PLA1 is derived fromAspergillus niger or Aspergillus oryzae.

The DNA sequence encoding the polypeptide to be produced may be operablylinked to appropriate DNA regulatory regions to ensure a high level ofexpression of said DNA sequence and preferably a high secretion level ofsaid polypeptide. If the polypeptide to be produced is native toAspergillus niger, its native secretion signal is preferably used.Alternatively, if the polypeptide to be produced is not native toAspergillus niger, a fusion construct is preferably made comprising theglucoamylase gene of Aspergillus niger fused to the heterologous gene tobe produced. According to a preferred embodiment of the invention, theregulatory regions of the Aspergillus oryzae alpha amylase gene areused. According to a more preferred embodiment of the invention, theregulatory regions of the A. niger glucoamylase gene are used. Accordingto a preferred embodiment of the invention, the alpha amylase secretionsignals are used. The DNA construct may also comprise a selectablemarker. Alternatively, the selectable marker may be present on a secondDNA construct. By way of example these markers include but are notlimited to amdS (acetamidase genes), auxotrophic marker genes such asargB, trpC, or pyrG and antibiotic resistance genes providing resistanceagainst e.g. phleomycin, hygromycin B or G418. Preferably, the markergene is the acetamidase gene from Aspergillus nidulans. More preferably,the acetamidase gene from Aspergillus nidulans is fused; to the gpdApromoter. Transformation methods of A. niger are well-known to theskilled person (Biotechnology of Filamentous fungi: Technology andProducts. (1992) Reed Publishing (USA); Chapter 6: Transformation pages113 to 156). The skilled person will recognize that successfultransformation of A. niger is not limited to the use of vectors,selection marker systems, promoters and transformation protocolsspecifically exemplified herein. After transformation, typically, the A.niger population is cultivated on a solid medium in a petri dish. Thetransformants selected after culture on solid medium are typicallycultivated in flask during three to seven days to check for expressionof the polypeptide.

Typically, for producing the polypeptide in the oxalate deficient A.niger strain in an industrial setting, a fed-batch fermentation processmay be used. At the end of the fermentation, the polypeptide can bepurified following techniques known to the skilled person. An example ofsuch a recovery technique is explained in the following. When thefermentation is stopped, the host must be killed. This is accomplishedby adding a killing-off agent at some specific temperature where thisagent can work effectively. For example, the killing-off agent may benatriumbenzoate or kaliumsorbate. Depending on the identity of thekilling-off agent chosen, the broth temperature is adjusted to thecorresponding working temperature of this agent, by using classicalcooling methods known to the skilled person. In the case of apolypeptide which is secreted into the fermentation medium, theseparation of the cell material from the polypeptide is for example asimple filtration process: the fermentation broth is filtrated using amembrane filter press equipped with a textile cloth (membrane filterpress and textile cloth can be obtained from Harborlite). To improve thefiltration performance, a suitable filter-aid can be used, together witha suitable pre-coat of the filter cloth.

To remove any remaining small particles, additional filtration steps canbe carried out, in such a way that a clear filtrate can be obtained. Thefiltrate can be polished filtered on filter plates with an average poresize of typically 1-10 micron. Several types of filter plates are knownto the skilled person and are here suitable. Subsequently, a germfiltration may be carried out using a filter with a pore size of about0.4 micrometer, to remove the major part of microorganisms. With thesetwo filtrations, a pre-coat may be used to improve the filtrationperformance. The filtrate may be then concentrated by ultrafiltration(UF) with a factor of typically 10-25. Several types of UF membranes aresuitable here. During UF molecules with a typical molecular weight ofless than a few thousands (depending also on the shape of the molecules)are removed from the filtrate. Thus, the relative amount of lowmolecular weight molecules to the polypeptide of interest may be reducedabout 10-25 times after UF. The duration of the UF varies depending onthe viscosity and filterability of the filtrate (which varies due tonatural variations in the raw materials). At that stage, theconcentration of the polypeptide present in the ultrafiltrate is usuallyhigh enough to proceed with the formulation of the polypeptide intoeither a liquid or a dry formulation depending on the applicationcontemplated.

A method was developed for obtaining oxalate deficient A. niger strainswhich are suitable for producing high yields of a polypeptide and whichcan be used as polypeptide producers in an industrial setting. Thepolypeptide may be homologous or heterologous for said A. niger. In caseof a heterologous polypeptide or enzyme, the wild type strain on whichthe method of the invention is applied may have been earlier transformedto express a gene coding for such polypeptide or enzyme as has beendescribed earlier in the description. Such oxalate deficient A. nigerstrains produce at least the amount of polypeptide the wild type strainsthey originate from produce under the same culture conditions.Preferably, the oxalate deficient A. niger strains produce morepolypeptide than the wild type strain they originate from under the sameculture conditions. According to another preferred embodiment, themutants produce at least the amount of polypeptide the A. niger strainCBS 513.88 produced under the same culture condition. More preferably,the mutants produce more polypeptide than the A. niger strain CBS 513.88produced under the same culture conditions.

This method comprises the following steps:

-   -   a) A. niger is subjected to UV irradiation,    -   b) MTP cultures of surviving colonies obtained in a) are        realized    -   c) a selection within the MTP cultures is performed in which        mutants are selected that produce no more than half the amount        of oxalate that the wild type strain they originate from        produces under the same culture conditions,    -   d) a second selection is performed within the mutants obtained        in step c) in which mutants are selected that produce at least        the amount of polypeptide the wild type strains they originate        from produce under the same culture conditions.

According to a preferred embodiment, the method comprises the followingsteps:

-   -   a) culture conditions are developed, which allow a production of        at least 15 mM oxalate in microtiterplates (MTP) or at least 30        mM oxalate in flask culture in the fermentation medium at the        end of fermentation,    -   b) A. niger is subjected to UV irradiation,    -   c) MTP cultures of surviving colonies obtained in b) are        realized under the culture conditions retained in a),    -   d) a selection within the MTP cultures is performed in which        mutants are selected that produce no more than half the amount        of oxalate that the wild type strain they originate from        produces under the same culture conditions,    -   e) a second selection is performed within the mutants obtained        in step d) in which mutants are selected that produce at least        the amount of polypeptide the wild type strains they originate        from produce under the same culture conditions.

According to another preferred embodiment, the method comprises thefollowing steps:

-   -   a) culture conditions are developed, which allow a production of        at least 15 mM oxalate in microtiterplates (MTP) or at least 30        mM oxalate in flask culture in the fermentation medium at the        end of fermentation,    -   b) A. niger conidiospores are subjected to UV irradiation,    -   c) MTP cultures of surviving colonies obtained in b) are        realized under the culture conditions retained in a),    -   d) a selection within the MTP cultures is performed in which        mutants are selected that produce no more than half the amount        of oxalate that the wild type strain they originate from        produces under the same culture conditions,    -   e) a second selection is performed within the mutants obtained        in step d) in which mutants are selected that produce at least        the amount of polypeptide the wild type strains they originate        from produce under the same culture conditions.

According to another preferred embodiment, the method comprises thefollowing steps:

-   -   a) A. niger is subjected to UV irradiation,    -   b) MTP cultures of surviving colonies obtained in a) are        realized,    -   c) a selection within the MTP cultures is performed in which        mutants are selected that produce at least the amount of        polypeptide the wild type strains they originate from produce        under the same culture conditions.    -   d) a second selection is performed within the mutants obtained        in c) in which mutants are selected that produce no more than        half the amount of oxalate that the wild type strain they        originate from produces under the same culture conditions,

Each step of these processes is characterized further below.

According to a preferred embodiment of the invention, in a first step,colonies of A. niger are first cultivated in a medium which allows aproduction of at least 30 mM oxalate in MTP or at least 100 mM oxalatein flask culture in the fermentation medium at the end of fermentation.The fermentation time should be at least 3 days. It is further apreferred embodiment of the method that the pH of this medium does notneed to be manually corrected. The pH of the medium of this step ismaintained between 3 and 7, preferably between 3.5 and 6.5, morepreferably between 4 and 6. Most preferably the pH of this medium ismaintained between pH 5 and 6. At such a pH value, the production ofoxalate is known to be high. The pH of the medium is preferably bufferedwith a solution of 2-[N-Morpholino]ethanesulfonic acid (MES) whoseconcentration is ranged between 0, 1 and 1 M, more preferably between0.15 and 0.55 M. Most preferably the MES concentration is 0.5 M. Anitrogen source is present in the medium of this step. Preferably thenitrogen source is a nitrogen source, which does not result in theacidification of the fermentation medium as a result of its uptake bythe cell. More preferably, the nitrogen source of the medium of thisstep is urea. According to a preferred embodiment of the presentinvention, the medium used in this step is the flask defined medium 2(FDM2) (see example 1). According to a preferred embodiment of thepresent invention, the A. niger strain used in this step is WT2 or theA. niger strain CBS 513.88 (see experimental information).

In a second step, A. niger is subjected to UV irradiation so that thesurvival percentage is ranged between 0.01% and 60%. Preferably, thesurvival percentage is ranged between 0.05% and 50%. More preferably,the survival percentage is 0.1%. It is well known to the skilled personthat conidiospores is the preferred material to mutagenize A. niger byphysical or chemical means. Mutants may however also be obtained frommycelium cells. The selection method described herein may be applied toselect mutants obtained from either conidiospores or mycelium cells.

In a third step, MTP cultures of the surviving population obtained in asecond step is performed during at least 3 days.

At the end of the MTP culture of the third step, mutants can be selectedin a fourth step on basis of their oxalate production (oxalate selectionstep). Preferably mutants are selected that produce no more than onethird of the amount of oxalate that the wild type strain they originatefrom produces under the same culture conditions. More preferably,mutants are selected that produce no more than one fifth of the amountof oxalate that the wild type strain they originate from produces underthe same culture conditions.

An assay to quantify the oxalate present in the medium that may be usedis described in the Examples. For practical reasons, the best mutants(the lowest oxalate producers) are retained for furthercharacterization. Preferably 5 to 50 mutants are retained for furthercharacterization. Typically, after 7 days in flask cultivation, it canbe checked that these selected mutants produce far less oxalate than thewild type strain: in the case described in FIG. 7, less than 5 mMoxalate is found in the fermentation medium of the mutants compared to40-45 mM for the wild type strain. After 7 days of fermentation, it canfurther be checked whether the medium is less acidified by the selectedmutants than by the wild type strain. It can also be checked bymeasurement of the biomass produced and by measurement of the residualglucose concentration at different intervals during fermentation thatthe low level of oxalate measured in the mutants is not the consequenceof either a poor growth and/or a poor metabolic activity of the selectedmutants.

A second selection step which can be applied to the mutants before orafter the oxalate selection step is the following: select mutants thatproduce at least the amount of polypeptide the wild type strains theyoriginate from produce under the same culture conditions. Preferably,the mutants produce more of a given polypeptide than the wild typestrains they originate from under the same culture conditions. Accordingto another preferred embodiment, the mutants produce at least the amountof a given polypeptide the A. niger strain CBS 513.88 produced under thesame culture condition. More preferably, the mutants produce more of agiven polypeptide than the A. niger strain CBS 513.88 under the sameculture conditions. To perform this last step, the mutants obtained inthe previous step and a wild type control are cultivated in liquidmedium for at least three days in a suitable medium. Preferably, thecultivation is performed during at least five days. At the end of theculture, the amount of the polypeptide produced may be determined usinga system for detection of said polypeptide as defined earlier on in theapplication. Preferably, if the polypeptide produced is an enzyme, theamount of active enzyme produced is determined by measurement of itsactivity in a model reaction (see examples).

An optional sixth step may be further applied to select for oxalatedeficient A. niger strains having an intracellular OAH activity which isdetectable as detected in a model reaction. Preferably, the modelreaction is the one described in experimental information in theExamples. More preferably, this step allows the selection of oxalatedeficient A. niger strains having an intracellular OAH activity, whichis ranged between 0.1 and 100% of the intracellular OAH activity of thewild type strain they originate from as detected in a model reaction,preferably between 0.5 and 90, more preferably between 0.5 and 80, evenmore preferably between 1 and 50, most preferably between 1 and 25 andeven most preferably between 1 and 10. According to another preferredembodiment, the oxalate deficient A. niger strains have an intracellularOAH activity, which is ranged between 0.1 and 100% of the intracellularOAH activity of the CBS 513.88 deposited strain as detected in a modelreaction. More preferably, the oxalate deficient A. niger strains of theinvention are strains having an intracellular OAH activity, which isranged between 1 and 90% of the intracellular OAH activity of the CBS513.88 deposited strain as detected in a model reaction.

The invention also relates to the use of an oxalate deficient A. nigerstrain for producing a given polypeptide. Accordingly, the inventionalso relates to a method for producing a given polypeptide wherein anoxalate deficient A. niger as defined in this application is used. Suchstrain produces at least the same amount of said polypeptide as the wildtype strain it originates from under the same culture conditions.Preferably, the strain produces more of said polypeptide than the wildtype it originates from under the same culture conditions. According toanother preferred embodiment, the strain produces at least the sameamount of said polypeptide or enzyme as the CBS 513.88 A. niger strainunder the same culture conditions. More preferably, the strain producesmore of said polypeptide or enzyme than the CBS 513.88 A. niger strainunder the same culture conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the oxalate assay standard curve. The measured opticaldensity is given as a function of the oxalate concentration present insolution.

FIG. 2 depicts the evolution of the pH of the culture supernatant ofwild type A. niger during fermentation in FDM1 medium with or without pHcorrection.

FIG. 3 depicts the average oxalate production obtained duringfermentation of the wild type A. niger in the FDM1 medium with orwithout pH correction.

FIG. 4 depicts the average oxalate production obtained duringfermentation of the wild type A. niger in the FDM1 medium as a functionof the MES concentration, with ammonium or urea as nitrogen source,without pH correction.

FIG. 5 depicts the average oxalate production obtained duringfermentation of the wild type A. niger in the FDM2 medium without pHcorrection.

FIG. 6 depicts the pH evolution during fermentation of wild type andsome selected oxalate deficient A. niger in the MDM1 medium.

FIG. 7 depicts the average alpha amylase produced after fermentation inthe FDM2 medium by the wild type and the 34 mutants as a function oftheir oxalate production.

FIG. 8 depicts the measured OAH activity in three oxalate deficient A.niger mutants and in the wild type.

FIG. 9 depicts the average oxalate production obtained during thefermentation of the wild type and oxalate deficient A. niger in the FDM2medium without pH correction.

FIG. 10 depicts the residual glucose concentration present duringfermentation of wild type and oxalate deficient A. niger in the FDM 2medium.

FIG. 11 depicts the pH evolution of culture supernatants of wild typeand oxalate deficient A. niger fermented in the FDM2 medium.

FIG. 12 depicts the evolution of the biomass produced duringfermentation of the wild type and oxalate deficient A. niger in the FDM2medium.

FIG. 13 depicts the production of a proline specific endoprotease in WT1and in FINAL (mutant 22) comprising the same estimated copy numbers ofthe gene coding for the proline specific endoprotease.

FIG. 14 depicts the production of phospholipase A1 in WT1 and in FINAL(mutant 22) in shake flask.

EXAMPLES

Experimental Information

Strains

WT 1: A. niger strain is used as a control for the level of oxalate, thelevel of a given polypeptide and the level of intracellular OAHactivity. This strain is deposited at the CBS Institute under thedeposit number CBS 513.88.

WT 2: WT 1 strain comprising several copies of an expression cassettecomprising the A. oryzae alpha-amylase gene integrated in the genome.This gene was already described elsewhere (Wirsel et al., (1989), Mol.Microbiol. 3:3-14). The original signal sequence coded by the A. oryzaealpha-amylase gene was replaced by the one of the glucoamylase gene fromA. niger. WT 2 was constructed and selected by techniques known topersons skilled in the art and described in EP 635 574 A1 and in WO98/46772.

OAH Activity Assay

Shake flask fermentations of different A. niger strains were performedas decribed hereafter. Cells were cultivated at 30° C., 170 rpm forthree days in 100 ml of OAH cultivation medium in 500 ml shake flaskswithout a baffle. The OAH medium is defined in Table 1 below. Then, thepH was shifted to 8 by addition of Na₂CO₃ and cells were cultivated foran additional 15 to 18 hours. Mycelium was harvested by filtration,washed with 0.9% (w/v) NaCl, frozen in liquid nitrogen and stored at−80° C. Frozen cells were disrupted in a mortar under liquid nitrogenand then suspended in the following extraction buffer: 100 mM MOPSbuffer pH 7.5 (MOPS=Morpholino propanesulfonic acid), 2 mM MnCl₂, 20 mMDTT, 5% sucrose. The suspension was centrifuged for 20 min. at 14,000r.p.m. at 4° C. in an Eppendorf centrifuge 5417R. 925 μl of the assaybuffer (assay buffer: 100 mM MOPS pH 7.5/2 mM Mn²⁺) was pre-heated at25° C. 25 μl of a 40 mM oxaloacetic solution was added to this preheatedmix. The oxaloacetic solution was prepared by dissolving 0.053 g ofoxaloacetic acid in 10 ml of the assay buffer. 50 μl of the suspensionobtained after centrifugation was added to the preheated mix. OAHactivity was determined according to the method described by Pedersen etal, 2000, Mol. Gen. Genet. 263:281-286. Briefly, oxaloacetate is used assubstrate. The enzyme activity was determined from the rate of decreaseof the absorbance (delta A/min) at 255 nm during 3 minutes with a timeinterval of 20 seconds and the absorption coefficient of oxaloacetate.The assay was carried out at 25° C. TABLE 1 OAH medium Trace MetalSolution ZnSO₄.7H₂O 0.143 g CuSO₄.5H₂O 0.025 g NiCl₂.6H₂O 0.005 gFeSO₄.7H₂O 0.138 g MnCl₂.4H₂O 0.060 g Water up to 10 ml OAH medium, pH =2.5 or 4.5, Sucrose 20 g KH₂PO₄ 1.5 g MgSO₄.7H₂O 1 g NaCl 1 g CaCl₂.2H₂O0.1 g NaNO₃ 15 g Trace Metal solution 0.5 ml (Adjust pH to 2.5 with HCl)Water up to 1 literProtein Assay

The protein content in the samples was determined according theCoomassie Plus Protein assay with Bovine Serum Albumin as a standardaccording to the manufacturer's instructions (Pierce, product number23236).

In Example 1, alpha amylase is given as an example of enzyme that can beproduced by an oxalate deficient A. niger strain at a level which is atleast the same as the one produced by the parental strain the mutantoriginate from under the same culture conditions.

Example 1 Method to Make Oxalate deficient Aspergillus niger Mutantswhich are High Polypeptide Producers

Oxalate Deficient A. niger Mutants were Made Starting from WT2.

1. Growth Media

Cultures were performed at 34° C., in 96-wells microtiter plates (MTPs)or 300 ml flasks with one baffle in a rotary shaker at a shaking speedof 220 rpm.

Flask precultures were inoculated with 17 000 spores per ml. 100 mlcultures were inoculated with 10 ml of preculture. TABLE 2 Flaskpreculture medium 1 (FPM1), pH 5.5 (all components are given in gramsper liter) Corn steep liquor 20 (Roquette-Fréres, France) Glucose.1H₂O22

TABLE 3 Flask defined medium 1 (FDM1), pH 6 (all components are given ingrams per liter) Glucose.1H₂O 82.5 Maldex 15 25 (Boom Mepel,Netherlands) Citric acid 2 NaH₂PO₄.1H₂O 4.5 KH₂PO₄ 9 (NH₄)₂SO₄ 15 ZnCl₂0.02 MnSO₄.1H₂O 0.1 CuSO₄.5H₂O 0.015 CoCl₂.6H₂O 0.015 MgSO₄.7H₂O 1CaCl₂.2H₂O 0.1 FeSO₄.7H₂O 0.3 MES* 30(*2-[N-Morpholino]ethanesulfonic acid)

Flask defined medium 2 (FDM2), pH 6: the FDM2 medium had the samecomposition as FDM1 except that 15 grams per liter urea are presentinstead of 15 grams per liter (NH₄)₂SO₄. This medium contained 100 gramsper liter MES instead of 30 grams. TABLE 4 Microtiter plate definedmedium 1 (MDM1), pH 6 (all components are given in grams per liter)Glucose.1H₂O 15 Citric acid 2 NaH₂PO₄.1H₂O 1.5 KH₂PO₄ 3 Urea 5 ZnCl₂0.02 MnSO₄.1H₂O 0.1 CuSO₄.5H₂O 0.015 CoCl₂.6H₂O 0.015 MgSO₄.7H₂O 1CaCl₂.2H₂O 0.1 FeSO₄.7H₂O 0.3 MES* 30(*2-[N-Morpholino]ethanesulfonic acid)2. Assay for Oxalate Detection in A. niger Culture Supernatant

A commercial kit available from Sigma diagnostics (Sigma. OXALATEdiagnostic kit, catalogus. nr. 591 year 2000-2001) was employed foroxalate quantification. The volumes recommended by the manufacturer weredownscaled to reach a final assay volume of 48 μl, the assay beingperformed in 384-wells MTPs. A Beckman Multimek 96 was employed for allliquid transfers and the absorbance was read at 550 nm in a BMGspectrofluorimeter. The Oxalate assay standard curve is given in FIG. 1(the optical density, OD, as a function of the oxalate concentration).In these conditions, the assay was found to be linear up to 2.5 mM.

3. Development of Cultivation Conditions to Maximize Oxalate Production

The wild-type strain employed throughout this section is WT 1.

The pH has been described as the most critical parameter for oxalateproduction. To achieve a high oxalate production, the pH of A. nigercultures should be maintained at a value close to 6 (Kubicek, C. P., etal, Appl. Environ. Microbiol. (1988) 54, 633-637; and Ruijter, G. J. G.,et al,. Microbiology (1999) 145, 2569-2576). Oxalate production sharplydecreases for pH values below 4 (Ruijter, G. J. G., van de Vondervoort,P. J. I., and Visser, J. 1999. Microbiology 145, 2569-2576). A pH closeto 6 can hardly be maintained in A. niger cultures, because of theproduction of several organic acids by the fungus. To test how criticalthe pH of the culture was in the FDM1 medium, triplicate flasks cultureswere performed with a wild-type A. niger strain, either with or withoutdaily manual pH correction by addition of sterile sodium hydroxyde. Apre-culture phase of 48 hours in FPM1 medium was performed before FDM1medium inoculation. In FDM1 medium, 0.15 M MES (30 g/L) was present tobuffer the medium acidification during A. niger growth.

As can be seen in FIG. 2, the buffer present in the medium was notsufficient to counterbalance the production of organic acids by A.niger, and FIG. 3 shows that the oxalate yield was greatly affected bythe pH of the culture. Cultures in which the pH was corrected yieldedabout 5 times more oxalate than the cultures in which the pH was notcorrected.

During the screening for oxalate deficient strains, A. niger was grownin conditions yielding a maximal oxalate production, so that oxalatedeficient strains could be selected and easily distinguished from astrain producing wild-type levels of oxalate. For practical reasons, amanual pH correction could not be an option to achieve a maximal oxalateproduction in the initial screening phase, when a huge number of mutantswere still under evaluation. To improve the level of oxalate productionwithout having the need to correct the pH of the cultures, twoparameters were tuned in the FDM1 medium, which were the MESconcentration in the medium and the nature of the nitrogen source.

As shown in FIG. 4, increasing the MES concentration and replacingammonium sulfate by urea had a major impact on the maximal oxalateconcentration, which could be reached in A. niger cultures without pHcorrection. In FIG. 5, the maximal oxalate concentration reached after 6or 7 days of fermentation depending on the composition of thefermentation medium is represented.

1M MES affected the growth of A. niger and an intermediate concentrationof 0.5 M MES was chosen. Thus the growth medium finally chosen for flaskcultivation during the screening was the FDM1 were the MES concentrationwas 0.5 M and where the ammonium sulfate was replaced by urea. From nowon, that medium will be referred to as FDM2.

FIG. 5 shows that in FDM2, the oxalate concentration reached wthout pHcorrection was equivalent to the oxalate concentration reached in FDM1with pH correction (compare with FIG. 3). So, there was no need for pHcorrection anymore.

4. First Selection: Oxalate Production

A. niger conidiospores were collected from WT 2 colonies sporulating onpotato dextrose agar (PDA) medium (Difco, POTATO DEXTROSE AGAR,cultivation medium, catalogus. nr. 213400, year 1996-1997). 10 ml of asuspension containing 4×10⁶ conidiospores per ml was subjected to UVirradiation at 254 nm (Sylvania, 15 Watts Black Light Blue tube, modelFT15T8/BLB) until an energy of 0.1783 J/cm² was received. A survival of0.1% of the initial number of colonies was obtained. The mutagenizedspores solution was plated on PDA medium and 10 000 survivors werepicked using a Genomic Solutions Flexys colony picker and further growninto 96 wells microtiter plates (MTP). These MTPs, called “masterplates”were incubated at 34° C. until a strong sporulation was apparent.

The masterplates were replica plated using the Genomic Solutions Flexyscolony picker into MTPs containing 40 μl of FPM1 and incubated for 48hours at 34° C. 170 μl of MDM1 was then added and the MTPs were furtherincubated for 7 days at 34° C.

The supernatant of the 10 000 individual cultures was assayed for thepresence of oxalate. In the cultivation conditions employed, the oxalateconcentration reached in cultures of the WT 1 and WT 2 strains was inthe range of 40 mM. The mutants for which the oxalate concentration inthe growth medium was below 12 mM were selected for a further selectionround. 255 mutants were retained. This second selection round was morestringent than the first one, so that it allowed to get rid of falsepositives.

The second mutant selection consisted of a quadruplicate MTP cultivationand assay for oxalate. The conditions employed were the same as the onesdescribed here above. Table 5, second column below lists the oxalateconcentration reached in the lowest producers amongst the mutants and inwild-type MTP cultures. TABLE 5 Mutants Average oxalate Average alphaconcentration amylase activity (mM) (U/ml)  1 1.51 3.5  2 6.34 3.7  310.61 6.3  4 13.25 6.9  5 4.46 5.2  6 9.18 6.7  7 10.41 4.7  8 11.47 3.9 9 2.09 3.4 10 3.23 4.3 11 4.05 5.9 12 5.87 3.1 13 7.36 4.4 14 9.82 3.315 2.5 7 16 1.28 3.1 17 2.86 4.6 18 2.39 5.1 19 5.71 5.8 20 4.19 4.5 212.25 6 22 0.78 5.4 23 0.5 3.9 24 1.38 4.3 25 6.42 6.6 26 7.16 5.2 272.28 3.9 28 2.33 4.5 29 8.15 5.5 30 3.21 5.3 31 3.7 4.6 32 1.84 4.4 331.87 4.8 34 8.54 4.4 WT 1 33.80 — WT 2 36.80 1.71 U/ml is the quantity of alpha amylase needed to convert 1 g solublestarch per hour into a product. The formation of this product is beingmeasured by following the absorption at 620 nm after addition of iode atpH 5.5 and at 30° C. The incubation time with iode is between 15 and 25minutes.

FIG. 6 shows that the selected mutant strains acidify less the MDM1growth medium upon growth compared to the wild-type strains.

5. Second Selection: Alpha Amylase Production

As a second selection step, the 34 mutants obtained in the formerparagraph were subsequently selected as to their capacities to producealpha amylase.

The 34 mutants and WT2 were grown the same way as in the formerparagraph, and characterized as to their alpha-amylase production.

The alpha-amylase activity present in culture supernatants wasdetermined using the alpha amylase assay kit from Megazyme (Megazyme,CERALPHA alpha amylase assay kit, catalogus. ref. K-CERA, year2000-2001). Table 5 third column lists the average alpha amylaseproduction detected in WT2 and in the 34 mutants.

FIG. 7 depicts the average production of alpha amylase as a function ofthe oxalate production of the 34 mutants and the wild type. It could beobserved in table 5, third column and in FIG. 7 that all the 34 mutantsproduced significantly more alpha-amylase than the wild-type strain theyoriginated from. All the oxalate mutants found at the former paragraphwere retained as mutants able to produce at least the same amount ofenzyme as the wild type they originate from under the same cultureconditions. Mutants 15, 19 and 22 were selected for further selection.

6. Third Selection: OAH Activity

As an additional selection, the intracellular OAH activity was measuredin the three mutants (15, 19, 22) selected at the former paragraph andas a control in WT1 and WT2. For some strains, measurements were madetwice (A, B) as indicated in FIG. 8. The test developed to measure OAHactivity is described in experimental data. Mutants 15 and 22 showed adetectable OAH activity (FIG. 8): approximatively 10 to 20% of the WT 1or WT2. Surprisingly mutant 19 showed a high OAH activity, which issimilar to the one of WT2. Surprisingly, these three oxalate deficientmutants still have a relative high OAH activity. Furthermore, they alsohave good enzyme production capacities.

Example 2 Characterisation of the A. niger Oxalate Deficient Mutants

1. Growth Media TABLE 6 FPM1 and FDM2 media; as defined in example 1.Flask preculture medium 2 (FPM2), pH 5.5 (all components are given ingrams per liter) Maltose.1H₂O 30 Casein hydrolysate 10 Yeast extract 5KH₂PO₄ 1 Tween 80 3 MgSO₄.7H₂O 0.5 ZnCl₂ 0.03 CaCl₂ 0.02 MnSO₄ 0.01FeSO₄.7H2O 0.32. Characterization of the A. niger Oxalate Deficient Mutants

Mutants 18, 22, 15, 23, 19, 33 were grown in the FDM2 medium, after 48hours of preculture phase in FPM1, and characterized as to their oxalateproduction, and several growth parameters (residual glucose, pH andbiomass formed). The results obtained with the FDM2 medium confirmed thelow level of oxalate production of the mutants compared to the wild-typestrains (FIG. 9).

The residual glucose present in the FDM2 medium during growth of wildtype and mutant strains was assayed using the Glucose assay kit fromSigma Diagnostics (Sigma, GLUCOSE diagnostic kit, catalogus nr. 510-A,year 2000-2001). As can be seen in FIG. 10, the glucose was almostcompletely consumed in some mutant cultures after 7 days of growth,suggesting the low oxalate level found in the selected mutant did notreflect a low metabolic activity. Only mutant 23 seemed to have areduced metabolic activity.

The pH of the cultures was also followed. As previously observed (seeexample 1), the acidification of the culture medium was less advanced inthe mutants than in wild-type cultures (See FIG. 11).

Finally, to ensure that the reduced oxalate production of the mutantswas not due to a poor growth, the biomass formation was followed byweighing the biomass dry weight formed in the cultures at variouscultivation times. Flasks were sacrificed at each time intervalconsidered and the total biomass dry weight content of the flask wasdetermined.

As can be seen in FIG. 12, the mutants showed various growth profilesbut tended to reach the same biomass level as the parental strain WT 2after 7 days of cultivation. Mutant 23 was the only one which shown alow level of biomass formation, but this level was still comparable tothe one reached by the wild-type strain WT 1 from which WT 2 originated.Mutant 23 was not retained as oxalate deficient mutant for furthercharacterization. The sporulation capacities of the mutants werevisually evaluated. It was found that the sporulation level of themutants was comparable to the one of the wild type strain they originatefrom. Only one mutant seemed to have lower sporulation capacities.

In the following examples, mutant 22 was used as oxalate deficient A.niger strain for producing different enzymes. This mutant was obtainedfrom WT2 and earlier on from WT1. In order to express other enzymes inthis mutant, all the copies of the alpha amylase gene were deletedaccording to the method described in EP 635 574 A, using the acetamidasegene as selection marker gene. This mutant empty of any foreign enzymeencoding gene would be named FINAL in the following examples.Subsequently, FINAL was transformed with expression construct comprisingthe gene coding for the corresponding enzyme to be expressed asdescribed in the following examples. In order to express specificenzymes in WT1, the expression constructs introduced in FINAL were alsointroduced in WT1 as described in the following examples. Copy numberwas checked. Mutant 22 was tested and compared to WT1 for the productionof a proline specific endoprotease and PLA1. Mutant 22 produced the sameamount of all enzymes tested as the WT1 it originates from under thesame culture conditions or even more.

Example 3 Comparison of the Production of a Proline SpecificEndoprotease in the WT1 and in FINAL Strains

The gene coding for the proline specific endoprotease, which has beenused has already been published elsewhere (WO 02/45524). In order toexpress the proline specific endoprotease described in WO 02/45524 inWT1 and in FINAL, the construct depicted in WO 02/45524 (pGBFIN11-EPO)was introduced in these strains by cotransformation as described in WO02/45524.

Transformants with similar estimated copy number were selected toperform shake flask experiments in 100 ml of the medium as described inEP 635 574 A1 at 34° C. and 170 rpm in an incubator shaker using a 500ml baffeled shake flask. After four days of fermentation, samples weretaken to determine the proline specific endoprotease activity. Theproteolytic activity of the proline specific endoprotease wasspectrophotometrically measured in time at pH 5 and about 37° C. usingZ-Gly(cine)-Pro(line)-pNA as a substrate. 1 U proline specificendoprotease is defined as the amount of enzyme which converts 1micromol Z-Gly(cine)-Pro(line)-pNA per min at pH 5 and at 37° C.

FIG. 13 shows that the proline specific endoprotease activity of the A.niger transformants with different estimated copy number is comprised ina range from 42 to 135 U/I. Strains with one estimated copy number havean activity of 42-46 U/I and correlates well with the activity of twoand three copy strains. We concluded that FINAL produces at least thesame amount of proline specific endoprotease as WT1 under the sameculture conditions.

Example 4 Comparison of Phospholipase A1 (PLA1) Production in WT1 and inFINAL Strains

We chose to express PLA1 from A. oryzae in WT1 and in FINAL. The geneencoding this enzyme has already been published (Watanabe I, et al,Biosci. Biotechnol. Biochem. (1999), Vol 63, numero 5, pages 820-826).This gene was cloned into pGBFIN11 using the same technique as describedin WO 02/045524 for the cloning of the proline specific endoproteasegene in pGBFIN11-EPO. This construct was introduced in these strains bycotransformation as described in WO 02/45524. Three independenttransformants of WT1 and FINAL were tested for PLA1 expression inshakeflasks. The transformants with similar estimated copy number werecultivated in 100 ml of the same medium as described in EP 635 574 A1 at34° C. and 170 rpm in an incubator shaker using a 500 ml baffeled shakeflask. After 2, 3, 4, 5 days of fermentation, samples were taken todetermine the PLA1 activity. To determine phospholipase PLA1 activityfrom Aspergillus niger (PLA1) spectrophotometrically, an artificialsubstrate is used: 1,2-dithiodioctanoyl phophatidylcholine (diC8,substrate). PLA1 hydrolyses the sulphide bond at the A1 position,dissociating thio-octanoïc acid. Thio-octanoic acid reacts with 4,4dithiopyridine (color reagent, 4-DTDP), forming 4-thiopyridone.4-Thiopyridone is in tautomeric equilibrium with 4-mercaptopyridine,which absorbs radiation having a wavelength of 334 nm. The extinctionchange at that wavelength is measured. One unit is the amount of enzymethat liberates of 1 nmol thio-octanoic acid from 1,2-dithiodioctanoylphosphatidylcholine per minute at 37° C. and pH 4.0.

The substrate solution is prepared by dissolving 1 g diC8 crystals per66 ml ethanol and add 264 ml acetate buffer. The acetate buffercomprises 0.1 M Acetate buffer pH 3.85 containing 0.2% Triton-X100. Thecolour reagent is a 11 mM 4,4-dithiodipyridine solution. It was preparedby weighting 5,0 mg 4,4-dithiodipyridine in a 2 ml eppendorf sample cupand dissolving in 1.00 ml ethanol. 1.00 ml of milli-Q water was added.The results are depicted in FIG. 14. It is shown that PLA1 activity intransformants of WT1 cultures decreased after 4-5 days. However, thePLA1 activity of transformants of FINAL accumulates during fermentationand no decrease in activity could be observed. We concluded that FINALproduces more PLA1 than the wild type counterpart it originates fromunder the same culture conditions.

1. An oxalate deficient A. niger strain for the production of a givenenzyme, wherein the oxalate deficient strain produces at least the sameamount of the enzyme as the wild type strain it originates from underthe same culture conditions.
 2. An oxalate deficient A. niger strainaccording to claim 1, wherein the oxalate deficient strain produces moreof the enzyme than the wild type strain it originates from under thesame culture conditions.
 3. An oxalate deficient strain according toclaim 1, wherein the oxalate deficient strain has an intracellular OAHactivity, which is between 1% and 25% of the intracellular OAH activityof the wild type strain it originates from as detected in a modelreaction.
 4. An oxalate deficient A. niger strain, characterized in thatwhen the strain has been transformed with an expression constructcomprising a gene coding for an enzyme, said strain produces at leastthe amount of the enzyme the wild type strain it originates from wouldproduce under the same culture conditions, when the wild type strain hasbeen transformed with the same expression construct as the oxalatedeficient strain.
 5. An oxalate deficient A. niger strain according toclaim 4, characterized in that the gene is an heterologous gene.
 6. Anoxalate deficient A. niger strain according to claim 1, wherein thestrain produces at least the amount of enzyme the A. niger strain CBS513.88 produced under the same culture condition, preferably more.
 7. Anoxalate deficient A. niger strain according to claim 1, wherein theenzyme is a fungal alpha amylase.
 8. An oxalate deficient A. nigerstrain according to claim 7, wherein the fungal alpha amylase is derivedfrom Aspergillus oryzae or A. niger.
 9. A method for obtaining oxalatedeficient A. niger strains which are suitable for producing at least theamount of enzyme the wild type strains they originate from produce underthe same culture conditions, said method comprises the following steps:a) A. niger is subjected to UV irradiation, b) MTP cultures of survivingcolonies obtained in a) are realized under the culture conditionsretained in a), c) a selection within the MTP cultures is performed inwhich mutants are selected that produce no more than half the amount ofoxalate that the wild type strain they originate from produces under thesame culture conditions, d) a second selection is performed within themutants obtained in step c) in which mutants are selected that produceat least the amount of enzyme the wild type strains they originate fromproduce under the same culture conditions.
 10. A method according toclaim 9, wherein the method comprises an additional step e) whereinmutant selected in step d) are further selected to have an intracellularOAH activity, which is between 1% and 25% of the intracellular OAHactivity of the wild type strain it originates from as detected in amodel reaction.
 11. A method of producing a given enzyme comprisingusing an oxalate deficient A. niger strain according to claim 1.